TW201717891A - Composition comprising ginseng derived exosome like vesicles for skin whitening - Google Patents

Abstract

Disclosed in the present disclosure are a composition for skin whitening containing ginseng-derived exosome-like vesicles and a method for preparing the ginseng-derived exosome-like vesicle. The ginseng-derived exosome-like vesicle may have a diameter of 20-500 nm and may be isolated from ginseng root. The composition containing the ginseng-derived exosome-like vesicles as an active ingredient has an effect of preventing, improving or treating a skin pigmentation disease such as melasma, freckle, lentigo, nevus, melanoma, etc. by effectively suppressing melanin production.

Description

Composition for skin whitening and containing ginseng-derived exosomal vesicles

SUMMARY OF THE INVENTION The present invention discloses a composition for skin whitening and containing ginseng-derived exosomal vesicles and a method for preparing the ginseng-derived exosomal vesicles.

Most animal cells have the ability to secrete extracellular vesicles of various sizes and compositions. These extracellular vesicles are present in all biological fluids, including blood, urine, saliva, and cultured medium (Loyer X, Vion AC, Tedgui A, Boulanger CM. Microvesicles as cell-cell). Messengers in cardiovascular diseases. Circ Res 2014; 114: 345-53; Ohno S, Ishikawa A, Kuroda M. Roles of exosomes and microvesicles in disease pathogenesis. Adv Drug Deliv Rev 2013; 65: 398-401).

Extracellular vesicles are membrane-structured vesicles having a diameter of about 20 nm to 5 microns. They vary in size and composition and contain a variety of species, such as exosomes (about 30-100 nm), ectosomes, microvesicles (about 100-1000 nm). , microparticles, etc.

Different types of extracellular vesicles are based on their source, diameter, density in sucrose, shape, rate of precipitation, lipid composition, protein labeling, sequestration type (ie, whether they are induced by signals or naturally generated). And so on to distinguish. For example, the micelles are irregular shapes of membrane vesicles having a size of about 100 to 1,000 nm. They are derived from the plasma membrane and are known to contain integrins, selectins, markers containing CD40 ligands, and lipids containing phospholipidylserines. Moreover, the exosomes are the smallest membrane vesicles having a cup shape and having a size of about 30 to 100 nanometers (<200 nm). They are derived from endosomes and are known to contain four transmembrane proteins such as CD63 and CD9, markers containing TSG101 and ESCRT, and cholesterol (cholesterols), sphingomyelins, nerves. Lipids of ceramides and phospholipids.

Extracellular vesicles reflect the state of the secretory-derived cells (donor cells), exhibit various biological activities depending on the cells from which they are secreted, and interact with cells at the same time as they transfer genetic material and proteins between cells. Has played an important role.

In plants, small vesicles are released into the extracellular space due to fusion between the plasma membrane and multivesicular bodies, and vesicles in the multivesicular body are observed in the extracellular space of various plant cells ( Marchant R, Peat A, Banbury GH. The ultrastructural basis of hyphal growth. New Phytol. 1967; 66: 623-629; Halperin W, Jensen WA. Ultrastructural changes during growth and embryogenesis in carrot cell cultures. J Ultrastruct Res . 18: 428-443; Marchant R, Robards AW. Membrane systems associated with the plasmalemma of plant cells. Ann Bot . 1968; 32: 457-471). Recently, it has also been reported that exosome-derived nanoparticles derived from plant cells and exosomes derived from mammalian cells are similar in composition of nano-sized vesicle structures and nanoparticles (An, Q). , Hückelhoven, R, Kogel, KH and van Bel, AJ (2006). Multivesicular bodies participate in a cell wall-associated defence response in barley leaves attacked by the pathogenic powdery mildew fungus. Cell Microbiol 8: 1009-1019; Regente, M , Pinedo, M, Elizalde, M and de la Canal, L (2012). Apoplastic exosome-like vesicles: a new way of protein secretion in plants? Plant Signal Behav 7: 544-546).

Exosomes are mainly used as biomarkers. However, techniques for using exosomes for specific purposes based on the intrinsic efficacy of exosomes have not yet been developed. In particular, the specific use of exocytic membrane vesicles derived from plant cells is poorly understood. For ginseng, although the skin whitening effect of the extract derived from ginseng or its components has been published, there is no report on the skin whitening effect of ginseng-derived exosomal membrane vesicles. A prior art on ginseng extract has been disclosed in Korean Patent Publication No. 10-2009-0130801.

[technical problem]

In one aspect, the present invention is directed to providing a composition for skin whitening comprising ginseng-derived exosomal vesicles instead of ginseng extract as an active ingredient.

In another aspect, the present disclosure is directed to a method for preparing a ginseng-derived exosomal vesicle.

[Technical Answers]

SUMMARY OF THE INVENTION The present invention provides a skin whitening composition comprising ginseng-derived exosomal vesicles as an active ingredient.

On the one hand, exocytic vesicles can be isolated from ginseng roots.

In another aspect, the exosomal vesicles can have a diameter of from 20 to 500 nanometers.

On the other hand, exosomal vesicles can be precipitated by ultracentrifugation of ginseng extracellular fluid at a gravitational acceleration multiple (xg) of 100,000 or higher.

In another aspect, the exosomal vesicles can have a buoyant density of from 1.00-1.20 grams per milliliter in iodixanol.

On the other hand, the active ingredient inhibits melanin production.

In another aspect, the active ingredient can prevent, ameliorate or treat a group selected from one or more of the following skin pigmentation diseases: liver spots, spots, freckles, sputum, melanoma, drug-induced Drug-induced pigmentation, post-inflammatory pigmentation, and dermatitis-induced pigmentation.

The present invention also provides a method for preparing a ginseng-derived exosomal vesicle, comprising: (1) a step of extruding ginseng to extract juice; (2) centrifuging the juice and removing the residue to obtain a supernatant. a step of supernatant; and (3) a step of obtaining an exosomal vesicle by ultracentrifuging the supernatant.

In one aspect, the centrifugation in step (2) can be carried out for 10-30 minutes at a multiple of 500-3,000 gravitational acceleration.

On the other hand, the ultracentrifugation in the step (3) can be carried out using a sucrose buffer density gradient and an iodixanol density gradient.

On the other hand, the ultracentrifugation in step (3) can be carried out for 1-6 hours at a multiple of 100,000-200,000 gravitational acceleration.

On the other hand, in the step (3), the exosomal vesicles can be obtained by ultracentrifuging the supernatant and separating the buoyant density in the iodixanol to a fraction of 1.00-1.20 g/ml.

[Beneficial effect]

In one aspect, the present invention provides an effect of supplying a skin whitening composition comprising ginseng-derived exocytic vesicles as an active ingredient.

In another aspect, the present invention provides an effect of supplying a method for preparing a ginseng-derived exosomal vesicle.

The details of the invention are described below.

SUMMARY OF THE INVENTION The present invention provides a composition for skin whitening comprising ginseng-derived exosomal vesicles as an active ingredient. SUMMARY OF THE INVENTION The present invention provides a composition for skin whitening comprising ginseng-derived exosomal vesicles, particularly exosomal vesicles derived from extracellular fluids of ginseng, rather than ginseng-derived extracts as active ingredient.

In an exemplary embodiment, the ginseng may be one or more selected from the group consisting of seeds, roots, stems, leaves, and fruits of a plant genus Panax . Specifically, the plant in the ginus genus (Genus Panax ) may be Korean ginseng (Panax ginseng), American ginseng (Panax quinquefolium), Panax notoginseng, Japan. Japanese ginseng (Panax japonicum), dwarf ginseng (Panax trifolium), Himalayan ginseng (Panax pseudoginseng), Vietnamese ginseng (Vietnamese ginseng) ) (Panax vietnamensis) and the like. There are no restrictions on its species, type, processing, growth environment, etc., including red ginseng, fresh ginseng, white ginseng, cultured ginseng or woody ginseng (woods) -grown ginseng), wild ginseng such as mountain-grown ginseng and cultured-root of wild ginseng.

In the context of the present invention, "exosomal vesicles" refer to nano-sized extracellular vesicles secreted by cells into the extracellular space. Vesicles include exosomes and are used in the broadest sense, including vesicles similar to exosomes in nano-sized vesicular structures and compositions.

As a result of fusion between the cell membrane and the multivesicular body, exosomal vesicles are secreted into the extracellular space by exocytosis. Exosomal vesicles are divided into internal and external by lipid bilayers, which are indirectly reflected by their constituents such as membrane lipids, membrane proteins, genetic material and cytosplasmic. Out of the nature and state of the cell. At the same time, exocytic vesicles serve as mediators of extracellular transporters for communication between cells and cells, and by combining with other cells and tissues, membrane components (membrane components) ), signaling RNA (mRNA), microRNA (miRNA), protein (growth hormones, cytokines), etc. are transferred to recipient cells.

In the context of the present invention, "ginseng-derived exosomal vesicles" refer to nanosized exosomal vesicles secreted by ginseng cells. Exosomal vesicles can be isolated from ecclesia extracellular fluids, or can be obtained physically or partially physically from existing tissues or cells.

In an exemplary embodiment, the exosomal vesicles can be isolated from the ginseng root.

In one aspect, the exosomal vesicles can be extracellular vesicles having a diameter of from 20 to 500 nanometers. Alternatively, the exosomal vesicles can be 20 nanometers or larger in diameter, 30 nanometers or larger, 40 nanometers or larger, 50 nanometers or larger, 60 nanometers or larger, 70 Nano or larger, 80 nm or larger, 90 nm or larger, or 100 nm or larger extracellular vesicles, and 500 nm or less, 450 nm or less, 400 Nai Meters or less, 350 nm or less, 300 nm or less, 250 nm or less, 200 nm or less, 150 nm or less, or 100 nm or less extracellular Vesicles.

On the other hand, exosomal vesicles can be precipitated by ultracentrifugation of ginseng extracellular fluid at a gravitational acceleration multiple of 100,000 or higher, especially at a multiple of 100,000-200,000 gravitational acceleration or a multiple of 100,000 gravitational acceleration.

In another aspect, the exosomal vesicles can have a buoyant density of 1.00-1.20 grams per milliliter, 1.03-1.20 grams per milliliter, or 1.06-1.20 grams per milliliter in iodixanol. Buoyancy density refers to the density measured by density gradient centrifugation.

In another aspect, the membrane composition of the exosomal vesicles can be chemically or physically modified to effectively perform the desired function in the target cells. For example, the membrane composition of exocytic vesicles can be chemically modified using a thiol group (-SH) or an amino group (-NH ₂ ), or via chemical bonding. An inducing substance, a fusogen, and polyethylene glycol are bound to the exosomal vesicles.

In an exemplary embodiment, the exocytic vesicles are permeable by a group selected from one or more of the following methods, including ultracentrifugation, differential centrifugation, balance. Equilibrium density centrifugation, density gradient, filtration, dialysis, and free-flow electrophoresis, but are not limited thereto.

Density gradients are the most commonly used methods when separating materials with different densities. According to the present invention, exocytic vesicles can be separated by a density gradient due to differences in density. As a specific example, a density gradient substance such as Ficoll, glycerol, sucrose, cesium chloride, iodixanol or the like can be used, but is not limited thereto. On the one hand, the density gradient can be used together with ultracentrifugation or the like. On the other hand, gel filtration or ultrafiltration can be used to isolate exosomal vesicles. On the other hand, dialysis can be used instead of filtration to remove small size molecules. On the other hand, free-flow electrophoresis can be used.

In an exemplary embodiment, the ginseng-derived exosomal vesicles can be obtained by the following methods: (1) a step of obtaining a juice containing ginseng extracellular fluid from ginseng; and (2) centrifuging the juice and removing The residue is subjected to a step of obtaining a supernatant; and (3) a step of ultracentrifuging the supernatant to obtain an exosomal vesicle.

In an exemplary embodiment, the juice in step (1) is a liquid extruded from ginseng and can be obtained by mechanical degradation, treatment with chemicals, and the like. In one aspect, the juice can be obtained by squeezing ginseng, and the squeezing can be suitably performed by a person of ordinary skill in accordance with methods known in the art.

In an exemplary embodiment, the centrifugation in step (2) can be performed for 10-30 minutes at a multiple of 500-3,000 gravitational acceleration. The speed or time of centrifugation can be changed by stages.

On the other hand, the ultracentrifugation in the step (3) can be carried out under a sucrose buffer solution density gradient and an iodixanol density gradient.

On the other hand, the ultracentrifugation in step (3) can be carried out for 1-6 hours at a multiple of 100,000-200,000 gravitational acceleration. The speed or time of centrifugation can be changed step by step.

On the other hand, the ultracentrifugation in the step (3) can be carried out at a gravity gradient of sucrose buffer solution and a density gradient of iodixanol at a magnification of 100,000 to 200,000 gravitational acceleration for 1-6 hours.

In another aspect, in step (3), the exosomal vesicles can be obtained by ultracentrifuging the supernatant and then separating the portion of the iodixanol having a buoyant density of from 1.00-1.20 g/ml. The separated fraction can be diluted with a buffer and then ultracentrifuged. Specifically, pelletized exosomal vesicles having a diameter of 20-500 nm can be separated by ultracentrifugation at 100,000 gravitational acceleration multiples for 2 hours.

In an exemplary embodiment, the active ingredient provides the effect of preventing, treating or ameliorating skin damage or skin disease associated with increased melanin either directly or indirectly. That is to say, the active ingredient provides an effect of preventing, treating or ameliorating a disease caused by excessive melanin production by effectively inhibiting melanin production.

In another aspect, the present invention provides a method of enhancing skin whitening comprising administering to a subject in need thereof a dose of a ginseng-derived exosomal vesicle capable of effectively enhancing skin whitening.

In another aspect, the present invention provides a method of inhibiting melanin production comprising administering to a subject in need thereof a dose of a ginseng-derived exosomal vesicle effective to inhibit melanin production.

In another aspect, the present invention provides a method for preventing, treating, and/or ameliorating skin damage or skin diseases directly or indirectly associated with increased melanin, and/or a disease caused by excessive melanin production, including A subject in need thereof is administered a dose of effective ginseng-derived exosomal vesicles which is effective in preventing, treating and/or ameliorating skin damage or skin diseases directly or indirectly associated with increased melanin, and/or by excessive A disease caused by melanin production.

In another aspect, the present invention provides ginseng-derived exosomal vesicles for enhancing skin whitening in a subject.

In another aspect, the present invention provides ginseng-derived exosomal vesicles for inhibiting melanin production in a subject.

In another aspect, the present invention provides ginseng-derived exosomal vesicles for use in preventing, treating, and/or ameliorating skin damage or skin diseases directly or indirectly associated with increased melanin, and/or caused by excessive melanin production. The disease.

In another aspect, the present invention provides the use of a composition for enhancing skin whitening in a subject and comprising a ginseng-derived exosomal vesicle composition.

In another aspect, the present invention provides a use for the preparation of a ginseng-derived exosomal vesicle composition for inhibiting melanin production in a subject.

In another aspect, the present invention provides the use of a composition comprising a ginseng-derived exosomal vesicle composition for preventing, treating and/or ameliorating skin damage or skin disease directly or indirectly associated with increased melanin , and / or diseases caused by excessive melanin production.

In an exemplary embodiment, the ginseng-derived exosomal vesicles can be used in a subject or administered to a subject in the form of a pharmaceutical composition, a cosmetic composition, or a food composition.

In an exemplary embodiment, the ginseng-derived exosomal vesicles can be used on the skin of a subject or administered to a subject for use on the skin.

In an exemplary embodiment, the disease caused by excessive melanin production may be one or more selected from the group consisting of: liver spots, spots, age spots, blemish, epidermal melanocyte lesions (epidermal) Melanocytic lesion), milk coffee spot (cafe's au lait macule), Becker's nevus, nevus spilus, freckles, dermal melanocytic lesion, Mongolian plaque Mongolian spot), nevus of Ota, acquired bilateral nevus of Ota-like macule, ito nevus of Ito, blue nevus, Melanocytic nevus, junctional nevus, compound nevus, intradermal nevus, halo nevus, congenital melanocytic nevus, history Spitz nevus, dysplastic nevus, melanoma, Lentigo maligna melanoma, superficial spreading melanoma, acal lentigin melanoma, nodular melanoma, pigmentary basal cell carcinoma (pigmented basal cell carcinoma), pigmented dermatofibromas, pigmented dermoid cyst, pigmented keloid, and pigmented keratoacanthomas.

In an exemplary embodiment, the composition may prevent, ameliorate or treat a group selected from one or more of the following: skin pigmentation, which occurs locally on the skin due to increased melanin production: liver spots, spots, Freckles, sputum, melanoma, drug-induced pigmentation, post-inflammatory pigmentation, and pigmentation caused by dermatitis.

In another aspect, the present disclosure provides a method for preventing, ameliorating, and/or treating a group selected from one or more of the following: skin pigmentation that occurs locally on the skin due to increased melanin production: Liver spots, spots, freckles, sputum, melanoma, drug-induced pigmentation, post-inflammatory pigmentation, and pigmentation caused by dermatitis, including effective ginseng-derived exosomes for subjects in need Vesicle dose.

In another aspect, the present invention provides a ginseng-derived exosomal vesicle for preventing, ameliorating and/or treating one or more of the following: skin pigmentation locally occurring on the skin due to increased melanin production; Group consisting of: liver spots, spots, freckles, sputum, melanoma, drug-induced pigmentation, post-inflammatory pigmentation, and pigmentation caused by dermatitis.

In another aspect, the present invention provides a use for the preparation of a composition comprising a ginseng-derived exosomal vesicle composition, which can be used to prevent, ameliorate and/or treat one or more selected from the group consisting of one or more of the following due to increased melanin production. Now a group of skin pigmentation on the skin: liver spots, spots, freckles, sputum, melanoma, drug-induced pigmentation, pigmentation after inflammation, and pigmentation caused by dermatitis.

In an exemplary embodiment, the composition can be a freeze-dried formulation. The composition may be a lyophilized formulation contained in a sealed packaging material or container, making it easy to use (ie, ready to use).

The present invention also provides a kit for skin whitening, inhibiting melanin production or for preventing, ameliorating or treating skin pigmentation, comprising: a freeze-dried combination containing ginseng-derived exosomal vesicles as an active ingredient And sterile water or pure water. The kit can be included in a sealed packaging material or container to make it easy to use (ready-to-use).

In an exemplary embodiment, the composition can be a pharmaceutical composition.

In addition to ginseng-derived exosomal vesicles, the pharmaceutical composition may also contain pharmaceutical adjuvants such as antiseptics, stabilizers, etting agents, emulsification promoters. The salt and/or buffer for controlling the osmotic pressure, and the like, and other therapeutically useful substances, and can be prepared into various preparations for oral or parenteral external use according to a usual method.

The preparation for oral administration may be, for example, a tablet, a pill, a hard or soft capsule, a liquid, a suspension, an emulsion, a syrup, Powder, dust, fine granule, granule, pellet, etc., and these preparations may contain, in addition to the active ingredient, a surfactant, a diluent (for example, lactose). (lactose), dextrose, sucrose, mannitol, sorbitol, cellulose, and glycine, lubricants (eg, cerium oxide, talc, stearic acid) And its magnesium or calcium salts as well as polyethylene glycol). Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methyl cellulose, Sodium carboxymethyl cellulose and polyvinylpyrrolidone, and may contain pharmaceutical additives such as disintegrants such as starch, agar or alginic acid or sodium thereof. Salts, absorbents, colorants, flavoring agents, sweeteners, etc. Tablets can be prepared by conventional mixing, granulating or coating methods.

The preparation for parenteral administration may be a preparation for transdermal administration such as an injection, a drop, an ointment, a lotion, a gel, a cream, a spray, a suspension, an emulsion. , suppository, patch, etc., but are not limited thereto.

The dosage of the active ingredient to be administered depends on the level of the person skilled in the art. The daily dose can vary depending on various factors such as the stage of the disease to be treated, age, health status, presence of complications, and the like. In one aspect, the composition can be administered at a daily dose of from 1 microgram/kg to 200 mg/kg, more specifically from 50 micrograms/kg to 50 mg/kg, 1-3 times a day. However, the dosage administered does not limit the scope of the invention in any way.

The pharmaceutical composition may be a preparation for external use on the skin. Formulations for external use on the skin include any preparation which can be externally applied to the skin, and various types of pharmaceutical preparations are also included.

In an exemplary embodiment, the composition can be a cosmetic composition.

In addition to ginseng-derived exosomal vesicles, the cosmetic composition may comprise functional additives and ingredients often included in cosmetic compositions. The functional additive may include a component selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, polypeptides, polysaccharides, sphingolipids, and seaweed extracts. In addition, it can also contain oils, fats, wetting agents, lubricants (emollient), surfactants, organic or inorganic pigments, organic powders, UV absorbers, preservatives, fungicides, antioxidants, plant extracts. , pH control agent, alcohol, coloring agent, fragrance, blood circulation promoter, cooling agent, antiperspirant, pure water, and the like.

The preparation of the cosmetic composition is not particularly limited and may be appropriately selected depending on the purpose. For example, it may be prepared as a group selected from one or more of the following: a skin lotion, a skin softener, a skin toner, an astringent, a lotion, an emulsion ( Milk lotion), moisturizing lotion, nourishing lotion, massaging cream, nourishing cream, moisturizing cream, hand cream, foundation ), essence, nourishing essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion and Body cleanser, but not limited to this.

When the preparation of the present invention is a paste, a cream or a gel, animal fiber, plant fiber, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, helium oxygen An alkyl polymer, bentonite, silica, talc, zinc oxide or the like can be used as the carrier component.

When the preparation of the present invention is a powder or a spray, lactose, talc, cerium oxide, aluminum hydroxide, calcium silicate, polyamide powder or the like can be used as a carrier component. . Especially when the formulation is a spray, it may also contain a propellant such as chlorofluorohydrocarbon, propane/butane or dimethyl ether.

When the preparation of the present invention is a solution or an emulsion, a solvent, a solubilizer or an emulsifier can be used as the carrier component. For example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol (propylene) can be used. Glycol), 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

When the preparation of the present invention is a suspension, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene dehydration. Polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, amaranth, tragacanth or the like can be used as a carrier component.

When the preparation of the present invention is a surfactant-containing detergent, an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic monoester, Isothionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate ), alkyl amidobetaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative ), ethoxylated glycerol fatty acid ester or the like can be used as a carrier component.

In an exemplary embodiment, the composition can be a food composition.

The food composition can be a liquid or solid preparation. For example, it may be in the form of various foods, beverages, gums, tea, vitamin mixtures, supplemental health foods, and the like, and may be formulated into powders, granules, tablets, capsules or beverages. Various formulations of the food compositions can be prepared by those skilled in the art without difficulty in admixing the active ingredient with ingredients which are conventionally employed in the art. A synergistic effect can be achieved when the active ingredient is used with other ingredients.

The liquid component contained in the food composition is not particularly limited except for the active ingredient. Various flavors, natural carbohydrates, and the like can be further included in the ordinary beverage. Natural carbohydrates may be monosaccharides, disaccharides such as glucose, fructose, etc., polysaccharides such as maltose, sucrose, etc., ordinary sugars such as dextrin, cyclodextrin (cyclodextrin) or the like, or a sugar alcohol such as xylitol, sorbitol, erythritol or the like. As a flavor, natural flavors (thaumatin, stevia extract (such as rebaudioside A, glycyrrhizin, etc.) or synthetic flavors (such as saccharin) can be used (for example, saccharin extract (such as rebaudioside A, glycyrrhizin, etc.) Saccharin), aspartame, etc., usually in an amount of from about 1 to about 20 grams, specifically from about 5 to about 12 grams per 100 milliliters of the composition of the invention.

In one aspect, the food composition may contain various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants, extenders (cheese, chocolate, etc.), pectic acid, and salts thereof. Alginic acid and its salts, organic acids, protective colloidal thickeners, pH control agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages, and the like. On the other hand, pulps for preparing natural juices and vegetable drinks can be included. These ingredients may be used singly or in combination. According to the present invention, about 0.001 to 20 parts of the additive is used per 100 parts by weight of the composition, but is not limited thereto.

Hereinafter, the present disclosure will be described in detail through embodiments. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited by the examples.

Example 1. Separation of ginseng-derived exosomes vesicles

(Step 1) First, a juice containing ginseng root extracellular fluid was prepared by extruding ginseng roots for 4 years.

(Step 2) The supernatant was centrifuged at 4 ° C and a gravity acceleration multiple of 10 for 10 minutes to obtain a supernatant. Then, the supernatant was centrifuged at 4 ° C and a magnification of 3,000 g for 20 minutes and the supernatant was taken.

(Step 3) Add 0.5 ml of 2.5 M sucrose, 1 ml of 0.8 M sucrose and 32 ml of the supernatant to remove impurities in a 35 ml ultracentrifuge tube, and centrifuge the tube at 4 ° C and 100,000 gravitational acceleration multiples. Centrifuge for 2 hours. After ultracentrifugation, the ginseng root exosomes vesicles are located between the 2.5M sucrose layer and the 0.8M sucrose layer due to their density. Therefore, the liquid layer containing the ginseng root exosomes vesicles can be separated by removing the solution from the upper side of the centrifuge tube.

(Step 4) The liquid layer containing the ginseng root exosomes vesicles separated was subjected to density gradient ultracentrifugation. Specifically, 0.42 ml of a separation layer containing ginseng root exosomal vesicles was mixed with 2.08 ml of 60% iodixanol. The resulting 2.5 ml of 50% iodixanol was added to a 12-mL ultracentrifuge tube. Then, after adding 2.5 ml of 35% iodixanol, 2.5 ml of 20% iodixanol and 2.5 ml of 150 mM sodium chloride / 20 mM hydroxyethylpiperazine ethanesulfonic acid (HEPES) to the tube, at 4 ° C Ultracentrifugation was carried out for 4 hours at a multiple of 200,000 gravitational acceleration. Thereafter, 1 ml of fraction was taken from the top of the tube, and fractionation 3 (1.08 g/ml) corresponding to the exocytic vesicle density was obtained by density measurement (see Fig. 1).

(Step 5) The obtained fraction 3 was diluted with HBS (HEPES-buffered saline) 60 ml, and added to a 70-ml ultracentrifuge tube. Then, granule-derived exosome-like vesicles were obtained by ultracentrifugation at 4 ° C and a magnification of 100,000 gravitational acceleration for 2 hours.

Test example 1. Transmitted electron microscopic analysis of ginseng-derived exosomes

The size and shape of the ginseng-derived exosomal vesicles obtained in Example 1 were analyzed by a transmission electron microscope (TEM).

Figure 2 shows a transmission electron microscope image of ginseng-derived exosomal vesicles. It was confirmed that the ginseng-derived exosomal vesicles have a diameter of 50-100 nm and are substantially spherical.

Test example 2. Ginsenoside-derived exocytosis vesicles 1 microgram / ML) treatment 1 week After MNT-1 Comparison of cell melanin production

The human melanoma cell line MNT-1 cells were placed in minimal essential medium (MEM) supplemented with 20% fetal bovine serum and cultured at 37 ° C under 5% carbon dioxide (CO 2 ). The cells were plated on 60-mm culture dishes, 1 × 105 cells per dish and allowed to stand overnight, and replaced with the same medium of 1 μg/ml of ginseng-derived exosomes obtained in Example 1. The medium. For comparison, the negative control group did not undergo any treatment. Fresh medium containing exocytic vesicles was replaced every 4 days and cultured for 8 days until they were filled with culture dishes. When the cells are fully grown, use 300 μl of lysis buffer (1% ethyl phenyl polyethylene glycol (NP-40), 50 mM Tris-HCl (Tris-HCl). The cells were treated with pH 7.5, 150 mM sodium chloride and collected in a microtube using a cell scraper. Then, after centrifugation at 15,000 rpm for 10 minutes, the precipitated particles were dissolved in 1 N sodium hydroxide (NaOH), and the absorbance was measured at a wavelength of 450 nm to compare the melanin content.

As a result, it was confirmed that the melanin content of the MNT-1 cells treated with the ginseng-derived exosomal vesicles was significantly lower than that of the MNT-1 cells not treated with the ginseng-derived exocytosis vesicles, as shown in FIG. (n = 6, *: p <0.05).

Test example 3. After treatment with various concentrations of ginseng-derived exosomal vesicles for 2 weeks, 20 Millijoule / Square centimeters ( mJ/cm ² Ultraviolet light B Light( UVB Irradiation 2 Comparison of melanin production by human melanocytes

Human primary melanocytes (Life Technologies, CA, USA) were placed at 37 ° C and 5% carbon dioxide in a human melanocyte growth supplement (HMGS; Gibco BRL, NY, USA). Incubation in supplemented M-254 medium (Gibco BRL, NY, USA). The cells were plated on a 100-mm culture dish, 2 x 10 ⁵ cells per dish and allowed to stand overnight, and irradiated twice with 20 mJ/cm 2 of ultraviolet B light for 24 hours at a time. Then, the cells were cultured for 2 weeks with the ginseng-derived exosomal vesicle medium obtained in Example 1, at a concentration of 0.1 μg/ml, 1 μg/ml, 5 μg/ml or 10 μg/day, respectively. The medium in milliliters was replaced once. For comparison, the positive control group was treated with 30 μM of thymol trimethoxycinnamate, 1 μM of Compound K or 2 parts per million (2 ppm) of fermented saponin for 2 weeks in the same manner, and the amount of melanin production thereof was measured. The negative control group was cultured in the same manner as melanocytes which were not treated with ultraviolet B light and melanocytes which were treated with ultraviolet B light but were not treated with the test substance. After 2 weeks, the cells were separated from the culture dishes and counted. The same number of cells were collected in a microtube, lysed with Protein Extraction Cell Lysis Buffer (RIPA) (Millipore, 20-188), and then centrifuged at 13,500 rpm for 30 minutes. The precipitated particles were dissolved in 1 N sodium hydroxide and photographed to compare colors. The absorbance was measured at a wavelength of 450 nm to compare the melanin content.

As a result, it was confirmed that when cells were treated with ginseng-derived exosomal vesicles at a concentration of 1 μg/ml or higher, the amount of melanin produced by ultraviolet B light irradiation was remarkably lowered as shown in FIG. (*: p < 0.05). The ginseng-derived exosomal vesicles obtained according to the present invention exhibit comparable skin whitening effects to Melosolv, Compound K and fermented saponin of AmorePacific. The amount of reduction in melanin content when cells were treated with the substance is listed in Table 1.

Table 1. Reduction of melanin content in human melanocytes irradiated twice with 20 joules per square centimeter of ultraviolet light B after treatment with the test substance (%)

Test example 4. Ginseng-derived exocytosis vesicles or ginseng juice liquid deal with 48 Human black after hours color Prime cell survival rate It Comparison

Human primary melanocytes (Life Technologies, CA, USA) were placed in M-254 medium (Gibco supplemented with human melanocyte growth additive (HMGS; Gibco BRL, NY, USA) at 37 ° C and 5% carbon dioxide. Cultured in BRL, NY, USA). After plating 0.7×10 ⁵ cells on a 24-well plate and allowing to stand overnight, the concentration of 1 μg/ml or 10 μg/ml was used to contain the ginseng-derived exocline obtained in Example 1. The medium of the vesicle (GEV), or the ginseng juice medium was replaced, or the medium without the former two was replaced (control group). After 48 hours of culture, cell viability was measured using a cell proliferation assay kit (WST-1 solution) (Roche Applied Science, Germany).

Results When the culture medium contained ginseng-derived exosomal vesicles (GEV) and ginseng juice at a concentration of 1 μg/ml, the cell viability was 92% and 86%, respectively, and when the medium concentration was 10 μg/ml, the ginseng derivative was obtained. Cell exocytosis (GEV) and ginseng juice, cell viability were 84% and 65%, respectively, compared to medium without any substance (control group), as shown in Figure 5 (*: p <0.05 ). Therefore, it was confirmed that ginseng-derived exosomal vesicles exhibited lower toxicity than ginseng juice when treated at the same concentration.

Test example 5. Using ginseng to derive exosomal vesicles or ginseng juice liquid deal with 2 week Rear, Take 20 Millijoule / Square centimeter Ultraviolet light B Light irradiation 2 Times It people class Melanocyte its melanin generate Comparison of quantities

Human primary melanocytes (Life Technologies, CA, USA) were placed in M-254 medium (Gibco supplemented with human melanocyte growth additive (HMGS; Gibco BRL, NY, USA) at 37 ° C and 5% carbon dioxide. Cultured in BRL, NY, USA). 2 × 10 ⁵ cells were plated on a 100-mm culture dish and allowed to stand overnight, and irradiated twice with 20 mJ/cm 2 of ultraviolet B light for 24 hours at a time. Then, the cells were cultured for 2 weeks with the ginseng-derived exosomal vesicle medium or ginseng juice medium obtained in Example 1, and replaced with a medium having a concentration of 1 μg/ml or 10 μg/ml, respectively, every 4 days. The control group was melanocytes cultured in the same manner but not treated with ultraviolet B light, and melanocytes treated with ultraviolet B light but not treated with the test substance. After 2 weeks, the cells were separated from the culture dishes and counted. The same number of cells were collected in a microtube, lysed with protein extraction cell lysis buffer (Millipore, 20-188), and then centrifuged at 13,500 rpm for 30 minutes. The precipitated particles were dissolved in 1 N sodium hydroxide and photographed to compare colors. The absorbance was measured at a wavelength of 450 nm to compare the melanin content.

As a result, when cells were treated with ginseng-derived exosomal vesicles (GEV) or ginseng juice, the amount of melanin production increased after ultraviolet B light treatment was remarkably lowered as shown in Table 2 and FIG. (*: p < 0.05, **: p < 0.01). However, considering that 10 μg/ml ginseng juice shows high cytotoxicity, it can be seen that the ginseng-derived exosomes vesicles of the present invention can more effectively reduce the increase of ultraviolet-induced melanin content and have low cytotoxicity. .

Table 2. Reduction of melanin content in human melanocytes irradiated with 20 mJ/m 2 of ultraviolet light B light after treatment with test substances (%)

Hereinafter, the contents of the present invention will be described in detail through the formulation examples. However, the following formulation examples are for illustrative purposes only and the scope of the present invention is not limited by the formulation examples.

[ preparation Instance 1] Soft capsule

50 mg of ginseng-derived exosomal vesicles, 80-140 mg of L-carnitine, 180 mg of soybean oil, 2 mg of palm oil, 8 mg of hydrogenated vegetable oil ( Hydrogenated vegetable oil), 4 mg of yellow beeswax and 6 mg of lecithin were mixed and each capsule was filled to 400 mg according to a usual method.

[ preparation Instance 2] tablet

50 mg of ginseng-derived exosomal vesicles, 200 mg of galactooligosaccharide, 60 mg of lactose and 140 mg of maltose were mixed and granulated with a fluidized-bed drier. 6 mg of the sugar ester was then tableted using a tablet press to prepare a tablet.

[ preparation Instance 3] Granule

50 mg of ginseng-derived exosomal vesicles, 250 mg of anhydrous crystalline glucose, and 550 mg of starch were mixed, and the mixture was granulated using a fluidized-bed granulator, and then filled in a small capsule to prepare granules. .

[ preparation Instance 4] Drink

A 50 mg ginseng-derived exosomal vesicle, 10 g of glucose, 0.6 g of citric acid, and 25 g of oligosaccharide syrup were mixed, 300 ml of pure water was added, and 200 ml of each bottle was filled to prepare a drink. After filling the bottle, the drink was sterilized at 130 ° C for 4-5 seconds.

[ preparation Instance 5] Lotion

The lotion was prepared according to the usual methods using the ingredients described in Table 3.

table 3.

[ preparation Instance 6] Cream

Creams were prepared according to the usual methods using the ingredients described in Table 4.

Table 4.

[ Formulation example 7] Bagged

The pouches were prepared according to the usual methods using the ingredients described in Table 5.

table 5.

Having thus described and described the exemplary embodiments, various changes in form and detail may be made by those skilled in the art without departing from the spirit and scope of the invention. Understood.

no

1 shows the process of isolating exosomoid vesicles from the obtained ginseng root juice according to an exemplary embodiment of the present invention.

2 shows a transmission electron microscope image of ginseng-derived exosomal vesicles obtained according to an exemplary embodiment of the present invention.

Figure 3 shows the amount of melanin production of MNT-1 cells treated with ginseng-derived exosomal vesicles obtained by an exemplary embodiment of the present invention.

Figure 4 compares ginseng-derived exosomal vesicle treatment or positive control substances obtained with an exemplary embodiment of the present invention (thymol trimethyl cinnamate (Melasolv) (Amore The amount of melanin produced by human melanocytes treated with representative skin whitening substances of the Pacific Ocean (Amore Pacific), Compound K and fermented saponin.

Figure 5 compares the viability of human melanocytes treated with ginseng-derived exosomal vesicles or treated with ginseng juice obtained in an exemplary embodiment of the present invention.

Figure 6 compares the amount of melanin production of human melanocytes treated with ginseng-derived exosomal vesicles or treated with ginseng juice obtained in an exemplary embodiment of the present invention.

Claims (12)

A composition for skin whitening comprising ginseng-derived exocytic vesicles as an active ingredient. The skin whitening composition of claim 1, wherein the exosome vesicles are isolated from ginseng roots. The skin lightening composition of claim 1, wherein the exosome vesicles have a diameter of from 20 to 500 nm. The skin whitening composition of claim 1, wherein the exosomal vesicles are precipitated by ultracentrifugation of ginseng extracellular fluid at a gravitational acceleration multiple of 100,000 or higher. The skin whitening composition according to claim 1, wherein the exosome vesicle has a buoyant density of 1.00-1.20 g/ml in iodixanol. The skin whitening composition according to claim 1, wherein the active ingredient inhibits melanin production. The composition for skin whitening according to claim 1, wherein the active ingredient prevents, ameliorates or treats a group selected from one or more of the following skin pigmentation diseases: liver spots, spots, freckles, moles, Melanoma, pigmentation caused by drugs, pigmentation after inflammation, and pigmentation caused by dermatitis. A method for producing a ginseng-derived exosomal vesicle according to any one of claims 1 to 7, which comprises: (1) a step of extruding ginseng to extract juice; (2) centrifuging the juice And removing the residue to obtain a supernatant; and (3) the step of ultracentrifuging the supernatant to obtain an exosomal vesicle. The method for producing a ginseng-derived exosomal vesicle according to claim 8, wherein the centrifugation described in the step (2) is carried out at a magnification of 500 to 3,000 gravitational acceleration for 10 to 30 minutes. The method for preparing a ginseng-derived exosomal vesicle according to claim 8, wherein the ultracentrifugation described in the step (3) is carried out under a sucrose cushion density gradient and an iodixanol density gradient. . The method for producing a ginseng-derived exosomal vesicle according to claim 8, wherein the ultracentrifugation described in the step (3) is carried out at a gravity acceleration multiple of 100,000 to 200,000 for 1-6 hours. The method for preparing a ginseng-derived exosomal vesicle according to claim 8, wherein in the step (3), the exosome vesicle is subjected to ultracentrifugation of the supernatant, and then the iodine is separated. The buoyancy density of the saxol is from 1.00 to 1.20 g/ml.